The diagnostically important molecules may be small molecules or macromolecules. The small molecules like mycotoxins such as aflatoxin B1, Ochratoxin A T-2 toxin are present in food and feed such as cereal grains, ground nuts, rice, peanuts, fodder as contaminants due to fungal infection. Human and animals are exposed to mycotoxins through ingestion of toxin-contaminated food and feed, inhalation or skin contact. The determination of mycotoxins in agricultural commodities is important because their consumption by man and animals causes mycotoxicosis. They also produce different biological effects such as acute toxicity, mutagenicity, carcinogenicity, tertogenicity etc (Morgan, M. R. A. Tetrahedron 45, 2237, 1989). The determination of the concentration of steroids such as estriol, dehydroepiandrosterone sulphate, cortisol, testosterone, hormones such as thyroxin, triiodothyronine and drugs such as methoxtrate present in biological fluid such as serum, plasma, urine in minute amounts have been accepted as one of the attractive indicators of several diseases and in pathological conditions (Methods of Hormone Radioimmunoassay Ed. Jaffe, B. M. and Behrman, H. R. 1979, Academic Press, New York).
The diagnostically important macromolecules like hormones such as thyroid stimulating hormones (TSH), adrenocortcotropic hormone (ACTH) or cancer markers such as acid phosphatase, ferritin, carcinoembroynic antigen are present in biological fluid such as serum, plasma in extremely minute amounts. These biologically important molecules have been widely accepted as the effective indicators of disease. Similarly, diagnosis of various infectious diseases also require detection of antigen such as hepatitis, malaria or detection of antibodies in cases of diseases like AIDS, amoebiosis (Methods of Enzymatic Analysis, Ed. By Bergmeyer, J. and Gross, vol. X, XI, 1983, Verlag chemie, Weinheim). These antigen or antibodies are present in biological fluid such as serum in extremely minute amounts and early detection sometimes is not possible. Some of these infectious diseases are detected by the present available methods only at a later stage when the concentration of antigen or antibody in biological fluid is increased.
Detection of these diagnostically important molecules requires highly sensitive assay system. Immunological assays have proven to be of great value for detection and quantification of numerous analytes in liquid samples. Because the results of immunological and other specific binding reactions are frequently not directly observable, various techniques have been devised for their indirect observation. Such techniques involve labeling of one of the members of the specific binding pair with a radioisotope, chromophore, fluorophore or enzyme label. Radiolabels, chromophores and fluorophores may be detected by the use of radiation detectors, spectrophotometers. Where members of a specific binding pair are tagged with an enzyme label, their presence may be detected by the enzymatic activation of a reaction system wherein a compound such as a dyestuff, is activated to produce a detectable signal. Such procedures are described in a number of articles and texts, an example of which is Reviews on Immunoassay Technology, Ed. S. B. Pal, Pub. Chapman and Hall, 1988.
Among the different immunoassays, enzyme labels tagged with specific binding pair are widely used. These assays are called enzyme immunoassay, commonly termed as ELISA. At present for small molecules competitive ELISA and for macromolecules sandwich ELISA method is widely used for their determination (Porstman, T. and Kiessig, S. T. Journal Immunological Methods 150, 5, 1992)
In competitive ELISA, sample or standard is added to a solid phase such as 96-well microtitre plate, tubes, beads coated with an antibody raised against small molecules to be determined. They are incubated after the addition of an enzyme, which is covalently linked with the small molecule at a temperature in the range of 4° C. to 37° C. for a period of 2 to 24 hrs. The analyte to be detected competes with a labeled reagent of the same analyte for a limited number of antibody binding sites. The amount of labeled antigen, which binds to the antibody, is inversely proportional to the amount of the unknown antigen in the sample.
The solid phases are washed with a buffer and substrate solution is added which gives colour due to the enzymatic activity of the enzyme conjugate bound to the antibody immobilized over solid phase. The intensity of the colour is inversely proportional to the concentration of small molecule present in the sample. Visualization of the intensity of the colour with naked eye in comparison with known concentration of small molecule gives an idea about the relative amount of small molecule present in the sample. The amount of colour developed may also be measured for quantitative determination by spectrophotometer or automatic microtitre plate reader. The measured absorbances are plotted against known concentration of small molecule to obtain a standard curve. Concentration of small molecule in samples is calculated from this standard curve by standard procedure.
In another assay, known as sandwich ELISA method, a solid surface such as 96-well microtitre plate, tubes are coated with monoclonal antibody obtained against macromolecules to be determined. Vacant sites are blocked with blocking protein such as casein, BSA. Standards or samples containing macromolecules are added to solid phase and incubated at a temperature in the range of 4 to 37° C. for a period of 2 to 24 hrs. The selected macromolecular antigen in the sample or standard binds to the receptor monoclonal antibody. The solid phase is washed and further incubated for a period of 2 to 24 hrs with a second polyclonal antibody (against macromolecules) covalently linked with an enzyme which is capable of binding to the bound antigen to form an immobilized reaction product. The solid phase is further washed. The label in the reaction product is detected which indicates the presence of the antigen in the sample. The concentration of macromolecule in samples is determined as described in above method.
These immunological detection methods as described above are widely used for detection of biologically important molecules commonly referred to as analytes. These methods are simple but have following drawbacks:    1. They require well-equipped laboratories and are designed for testing samples in batches.    2. The presence of mostly long incubation period makes the method elongated and time consuming.    3. The detection limit of the method is low, making the detection of analytes present in extremely minute amount very difficult.    4. These assays are not suitable for on-site testing or for use under field conditions.
Dot-immunobinding assays introduced as an alternative to ELISA for detection of antigen or antibody (Hawkes, R., et. al. Analytical Biochemistry 119, 142, 1982) have brought a great revolution in the field of diagnostic. These methods commonly referred to as membrane-based assays are similar to ELISA and only difference is that here membrane are used as immunosorbent instead of microtitre plate or tubes. They also have competitive methods for small molecules and non-competitive or sandwich methods for macromolecules. Here monoclonal antibody that is capable of specifically binding to the target substance is immobilized over the membrane. In the assay, the sample to be tested is applied to the reaction membrane. If the target analyte is present in the sample, it will bind to the immobilized receptor. Typically after incubation step the sample was separated from the solid phase, which was then washed and incubated with a solution of additional polyclonal antibodies covalently, labeled with enzyme. After incubation, the unbound-labeled antibody was separated from the solid phase and the amount of labeled antibody bound to the solid phase was determined. These methods are simple, colour is visible over white background of the membrane. The method has following drawbacks:    1. The detection limit of the method is low and is in the range of 500 to 2000 pg making the detection of antigen or antibody in test samples difficult.    2. The long incubation period makes the method time consuming.    3. The low detection limit of the method has limited its application for detection of antigen or antibody, which are present in extremely minute amount.    4. Membranes are delicate in nature and their handling becomes difficult.
Detection of analyte present in low concentration requires sample concentration or longer incubation time to generate sufficient signal for accurate estimation of analytes. However, because of the low sensitivity of membrane-based assays and non-specific interference, interpretation of the results may not be accurate. Application of enzyme amplification step has been proved to be very efficient in not only increasing the sensitivity but also reducing the assay time (Bates, Trends in Biotechnology. 5, 204, 1987). In these procedures, the solid phase bound primary enzyme is linked catalytically to an additional system, which not only amplifies the signal but also increases the sensitivity.
Bobrow et al., (Journal of Immunological Methods, 125, 279, 1989; Ibid. 137, 103, 1991) described a signal amplification system called ‘Catalyze Reporter Deposition’ (CARD) method to improve detection limit in immunoassays using rabbit IgG as test analyte. In this method, diluted solution of antigen (rabbit IgG) at different concentration was applied over nitrocellulose membrane strip as dots. It was dried, blocked with 5% casein and incubated with double antibody-peroxidase conjugate. It was again incubated with biotin-tyramine conjugate containing 0.004% hydrogen peroxide. Membrane bound peroxidase catalyzes phenolic portion of biotin-tyramine conjugate which deposits on to the surface of membrane. The deposited biotin is then reacted with streptavidin labeled enzyme thereby resulting in deposition of enzymes. The net effect is that a single HRP label is surrounded by many peroxidase molecules. The membrane is incubated with substrate solution and due to enzymatic activity colour develops over the membrane as dots. The intensity of colour is directly proportional to the amount of colour developed. The method also called ‘Tyramide Signal Amplification’ is flexible and can be applied as an additional step after conventional Dot-ELISA. In this method, due to deposition of additional enzyme results in amplification and thereby improving the detection limit by more than 25-fold. However, this method has following disadvantages:    1. The detection limit of the method with different substrate solution depending on the enzyme label used for streptavidin is in the range of 80 to 1 pg.    2. The increased in sensitivity is only 30-fold.    3. Long incubation periods required makes the method time consuming.
Recently, the applicants have developed a novel signal amplification method based on catalyzed reporter deposition. The method termed Super-CARD method utilizes synthesized electron rich proteins having multiple copies of phenolic group as blocking agents. After completion of conventional assay, the solid phase bound HRP oxidises the added labeled substrate, which deposits onto the solid phase. This deposition is markedly increased in the presence of immobilized electron rich proteins, which not only amplifies the signal but also increases the sensitivity. The high specificity of the amplification reaction avoids the generation of any false positive signal. Direct comparison with existing CARD methods demonstrates approximately 1.6×104-fold enhancement in detection sensitivity which is much higher than that of any other existing methods (Indian Pat. No. 1996/DEL/97; 1989/DEL/97; 1991/DEL/97 and published in Journal of Immunological Methods 227, 31-39, 1999; Ibid. 230, 71-86, 1999). The method offers several advantages:    1. The method is simple and easy to implement.    2. The novel protein conjugates used as blocking agents can be prepared from commercially available inexpensive proteins and chemicals.    3. The same reagents used in CARD amplification can be used.
As the membranes are very delicate and difficult to handle, numerous assay devices, in various configurations were developed for wider use under field conditions.
The dipstick was first to be introduced, generally uses a plastic strip with membrane containing immobilized antibody attached at one end for dipping into a solution either containing or suspected of containing the analyte of interest. When incubated, analyte present in the sample binds to antibody immobilized over membrane. The extent to which the analyte becomes bound to that zone can be determined with the aid of labeled reagents. Typically, the user determines the concentration of the analyte by comparing the colour on the membrane to the colour on an external calibrator, such as a series of coloured plates that are printed on a label. The colour of each plate is associated with a particular concentration of the analyte. The colour on the plate that most closely approximates the colour on the dipstick provides the user with an approximate concentration of the analyte in the test samples. The method has however several drawbacks:    1. Method is time consuming and often requires a number of manipulative steps, for example, the addition and incubation of assay reagents.    2. It is difficult to match the colour of the plates with the colour on the dipstick.    3. Only one sample can be analyzed by one dipstick.    4. The colour on the plates would not fade in proportion to the adverse conditions affecting the colour on the dipstick. Thus, for a particular set of reaction conditions, the comparison of results with the colour on the plates will not give accurate result.
The Immunochromatographic test strip device constitutes an improvement over the simple dipstick. This class of devices has an absorbent strip immobilized with receptor (antibody) near the center of a typically rectangular chromatography medium, e.g. filter paper, membrane and having an end portion for contacting a test solution. The strip having a length and width is capable of conveying fluids in a fluid flow direction generally parallel to the length of the strip. They generally exhibits improved sensitivity in analyte detection relative to that of simple dipstick devices by virtue of the analyte concentrating effect achieved by the flow of sample containing the analyte past an immobilized analyte binding zone. A sample that is suspected of containing the analyte of interest is placed at or near one end of a membrane strip followed by the labeled reagent. The label reagent is an second antibody different from the first antibody yet it also binds with specificity to the analyte, is prepared separately and bound to a detectable marker substance to prepare a marker-second antibody complex. The maker-second antibody complex can be premixed with sample prior to addition to the strip or it can be added substantially simultaneously with the sample or it can be added after sample addition. The mixture is allowed to be carried to the opposite end of the membrane strip by a liquid phase that traverses the membrane strip by capillary action. While traversing the membrane strip, if the sample contains analyte it binds to the receptor (either mobile or stationary phase) and the marker is also captured by the trap yielding a complex of (marker)-(second antibody)-(analyte)-(first antibody). Because the marker is detectable, the presence of the marker can be detected by the naked eye, i.e. by means of colour contrasting with the chromatographic medium. Therefore, a coloured mark or the like will be left by the marker at the site at which the first antibody was affixed and thereby it is possible to easily confirm the presence (or absence) of the analyte. At present, many such in vitro diagnostic kits based on immunochromatography are known and available commercially. The methods are simple, less time consuming however has several drawbacks:    1. The method can only be used for determining the presence or absence of substance of interest or of clinical significance. No quantification of the analyte is possible.    2. The materials and dimensions influence the evenness of the flow of the detecting molecules through the assay. If the flow of liquid is too fast, the detectable molecules are left behind and are not accurately detected by the assay.    3. Only single sample can be analyzed with one assay device.
Flow-through devices, which overcome some of the disadvantages of the Dip-Stick and Immunochromatographic test strip devices were developed using membrane such as nitrocellulose, glass fibre, polyester, cellulose nitrate, polyester, nylon pre-coated with an antigen. These devices utilizes flow of fluid in a direction which is primarily transverse to the plane of the membrane A solution containing the target analyte is drawn through the entire membrane area by capillary action of the adsorbent material located adjacent to the membrane. Absorbent material such as cellulose acetate, filter paper, porous polyethylene is capable of absorbing liquid sample in substantially greater amount than that applied during one test. The absorbent body provides a means to collect the sample by providing uniform suction to deliver the sample through the reaction membrane down into the adsorbent body. Thus, the adsorbent body also acts as a reservoir to hold the sample, and various reagents. Samples containing target analyte and reference standards is applied to different areas onto the membrane surface and absorbency of the absorbent will draw the liquid of the sample. Thereafter, signal producing systems capable of generating a detectable visual change on the surface attached to a antibody having binding specificity for the target analyte is drawn through the membrane surface. When the colour producing system is used, antibody conjugated to horseradish peroxidase is exposed to the membrane surface. Subsequent exposure of the membrane to 4-Chloro-1-Naphthol substrate results in deposition of a dark blue dye on the membrane surface due to enzymatic activity. High contrast between the dyed and undyed portions of the membrane surface allows for detection of analyte. Visualization of the intensity of the colour in comparison with known concentration of reference standard gives an idea about the amount of antigen present in the sample. The method is simple and test can be performed under field conditions and results obtained usually under 10 minutes.
Several analytical devices based on Flow-through principle have been developed and described in patents which employs a membrane immunoadsorbent in combination with an absorbent pad. The absorbent body, which constitutes the fluid-receiving zone in these devices, can either be in non-continuous contact with the membrane containing immobilized antibody (U.S. Pat. No. 4,246,339) or in continuous contact with membrane (U.S. Pat. Nos. 4,366,241, 4,446,232, 4,632,901 and 4,727,019). Devices in which absorbent body is not in direct contact with the membrane permit the solution containing sample and/or labeled reagents grater contact with membrane before flow of solution to absorbent body takes place. Such non-continuous contact devices are more efficient at utilization of sample and labeled reagents however they require physical motion by assayist to bring in the flow of liquid. This step can bring about error in the assay. The continuous contact devices are less efficient in utilization of costly-labeled reagents. Thus, a reagent volume substantially greater than the void volume of the membrane is required to ensure that the entire membrane has been contacted with the solution containing reagents.
In both of these types of devices the membrane and absorbent body are contained in a plastic housing having a top member and a bottom member joined together under compression to hold the membrane and absorbent body in place and in contact with each other. In a recent U.S. Pat. No. 5,885,526 to Chu (March 1999), analytical device has been slightly modified by sealing reaction membrane and absorbent body with water insoluble adhesive. Liquid sample is applied to the pad by various techniques, and the sample drawn through the entire membrane area by capillary action of the absorbent pad. The rate and path of fluid flow in assay device has great effect on assay results. A number of devices have been described in the prior art which use surfaces with specifically arranged geometric elements to control the path and the rate of fluid flow. Devices such as are described in U.S. Pat. Nos. 4,426,451 and 5,922,615 utilize an arrangement in which a membrane is placed between smooth surfaced planer sheets of a non-absorbent body in order to contain a fluid within the membrane. Devices such as are described in U.S. Pat. Nos. 4,233,029 and 4,310,399 use geometric arrangements of capillary channels to modulate the flow of fluid, such that fluid is directed to flow in regular geometric patterns and at controlled rates. Detection of analyte present in low concentration requires sample concentration. This has been achieved in some analytical devices (U.S. Pat. No. 4,818,677) by having high capacity adsorbent body beneath the reaction membrane, which draws larger volume of sample, added to the top of the membrane. However, because of the non-specific interference, interpretation of the results may not be accurate. The major drawbacks of the developed Flow through devices till date are as follows:    1. The low detection limit of analytical device has limited its application for detection of antigen or antibody, which are present in extremely minute amount.    2. Analytical devices are assembled individually making the manufacturing process complicated and costly.    3. The use of insufficient compression to hold the membrane and absorbent body tends sample to flow laterally during assay leading to inaccuracy in result.    4. Requires application of pressure to force liquid from membrane to absorbent layer.    5. Application of signal amplification step difficult.